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DR ANTHONY MELVIN CRASTO, Born in Mumbai in 1964 and graduated from Mumbai University, Completed his Ph.D from ICT, 1991,Matunga, Mumbai, India, in Organic Chemistry, The thesis topic was Synthesis of Novel Pyrethroid Analogues, Currently he is working with GLENMARK PHARMACEUTICALS LTD, Research Centre as Principal Scientist, Process Research (bulk actives) at Mahape, Navi Mumbai, India. Total Industry exp 30 plus yrs, Prior to joining Glenmark, he has worked with major multinationals like Hoechst Marion Roussel, now Sanofi, Searle India Ltd, now RPG lifesciences, etc. He has worked with notable scientists like Dr K Nagarajan, Dr Ralph Stapel, Prof S Seshadri, Dr T.V. Radhakrishnan and Dr B. K. Kulkarni, etc, He did custom synthesis for major multinationals in his career like BASF, Novartis, Sanofi, etc., He has worked in Discovery, Natural products, Bulk drugs, Generics, Intermediates, Fine chemicals, Neutraceuticals, GMP, Scaleups, etc, he is now helping millions, has 9 million plus hits on Google on all Organic chemistry websites. His friends call him Open superstar worlddrugtracker. His New Drug Approvals, Green Chemistry International, All about drugs, Eurekamoments, Organic spectroscopy international,
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Various HPLC conditions can be used to determine the chemical purity of the compounds disclosed herein. One such example is disclosed above in relation to the thermodynamic aqueous solubility studies. Another example is disclosed below.

Under these conditions, the purity of 4, RP-4, and SP-4 was determined to be ˜99.6, ˜99%, and ˜99.5%, respectively. It is noted that higher purities can be realized by optimizing the methods disclosed above.

Inspection of the XRPD diffractograms shows that the two crystalline single diastereoisomers gave clearly different XRPD patterns. Additionally, there was a clear difference in the melting point of the two crystalline diastereoisomers, with RP-4 having a considerably higher onset than SP-4 (136° C. vs. 94° C.).

Example 29Additional Separation Methods

The following SFC separation (conditions listed below) yielded adequate separation of a mixture of the diastereomers, RP-4 and SP-4.

Preparative Method:

Analytical Method:

Chiralpak AS-H (2 × 25 cm) SN# 07-8656

Chiralpak AS-H (25 ×

0.46 cm)

20% methanol/CO2 (100 bar)

20% methanol/CO2 (100 bar)

50 ml/min, 220 nm.

3 ml/min, 220 nm.

Conc.: 260 mg/30 ml methanol,

inj vol.: 1.5 ml

The following SFC separation (conditions listed below) yielded adequate separation of a mixture of the diastereomers, RP-4 and SP-4.

Preparative Method:

Analytical Method:

Chiralpak IA(2 × 15 cm) 802091

Chiralpak IA(15 × 0.46 cm)

30% isopropanol(0.1% DEA)/CO2,

40% methanol(DEA)/CO2, 100 bar

100 bar

60 mL/min, 220 nm.

3 mL/min, 220 nm.

inj vol.: 2 mL, 20 mg/mL methanol

TABLE 16

Summary of results from the batch characterization of RP-4, 4, and SP-4.

Analysis

RP-4

4

SP-4

Proton NMR

Single diastereoisomer

1:1 Mixture of

Single diastereoisomer

diastereoisomers

XRPD

Crystalline – different

Amorphous

Crystalline – different

DSC

from SP-4

Endotherm; 59° C.

from RP-4

Endotherm; melt – 136° C.

Endotherm; melt – 94° C.

TGA

No wt loss,

No wt loss, decomposition

No wt loss,

decomposition >240° C.

>240° C.

decomposition >240° C.

IR

See above

See above

See above

Aq Solubility

1.58

6.11

5.65

(mg · ml−1)

HPLC Purity

96.9%

99.6%

99.5%

40° C./75% RH

No form change

Deliquescence inside 1.5 h

Deliquescence inside 4.5 h

25° C./53% RH

—

Deliquescence

No form change

GVS

Non-hygroscopic up to 90%

—

Non-hygroscopic up to 60%

RH

RH

Example 27Thermodynamic Aqueous Solubility

Aqueous solubility was determined by suspending a sufficient amount of compound in water to give a maximum final concentration of ≧10 mg.ml−1 of the parent free-form of the compound. The suspension was equilibrated at 25° C. for 24 hours then the pH was measured. The suspension was then filtered through a glass fiber C filter into a 96 well plate. The filtrate was then diluted by a factor of 101. Quantitation was by HPLC with reference to a standard solution of approximately 0.1 mg.ml−1 in DMSO. Different volumes of the standard, diluted and undiluted sample solutions were injected. The solubility was calculated using the peak areas determined by integration of the peak found at the same retention time as the principal peak in the standard injection.

TABLE 14

HPLC Method Parameters for Solubility Measurements

Type of method:

Reverse phase with gradient elution

Column:

Phenomenex Luna, C18 (2) 5 μm 50 × 4.6 mm

Column Temperature

25

(° C.):

Standard Injections (μl):

1, 2, 3, 5, 7, 10

Test Injections (μl):

1, 2, 3, 10, 20, 50

Detection:

260, 80

Wavelength,

Bandwidth (nm):

Flow Rate (ml · min−1):

2

Phase A:

0.1% TFA in water

Phase B:

0.085% TFA in acetonitrile

Time (min)

% Phase A

% Phase B

Timetable:

0.0

95

5

1.0

80

20

2.3

5

95

3.3

5

95

3.5

95

5

4.4

95

5

[0306]

Analysis was performed under the above-noted conditions on an Agilent HP1100 series system equipped with a diode array detector and using ChemStation software vB.02.01-SR1.

Blank plasma samples used for matrix effect (ME) assessment and for the preparation of calibration and control samples were obtained from citrated blood (1,850 × g, 10 min, +4°C, Beckman J6B centrifuge) collected from Vaquez disease patients on the occasion of their regular phlebotomy.

The blank plasma used for the preparation of the calibration and quality control (QC) samples was acidified with 10% FA (50 μl of 10% FA added to 950 μl of plasma). The acidification of plasma aims at preventing the conversion of TVR to its epimer VRT-127394 that occurs in vivo and in vitro. (Tibotec-Janssen, personal communication).

(1) a nucleophilic reagent as NaSCN, the phase transfer catalyst is TBAB

The compound (product of Example 1, step (a)) is represented by the formula 3-1 is dissolved in dichloromethane (20ml) was added TBAB (2.8mmol), the NaSCN (35mmol) in water (2.0ml) was added dropwise It was added to the reaction solution. Dropping was completed, stirring was continued for 60 minutes, the solid was removed by filtration. The filtrate was washed with water, add MgSO 4 dried for 24 hours. Filtered, and the filtrate was evaporated under reduced pressure, to obtain a compound of formula 3-2 as (where X = SCN).

The compound (product of Example 1, step (a)) is represented by the formula 3-1 is dissolved in ethyl acetate (20ml) was added 18-crown -6 (2.8mmol), the NaSCN (35mmol) was added to the above the reaction mixture. Dropping was completed, stirring was continued for 60 minutes, the solid was removed by filtration. The filtrate was washed with water, add MgSO 4 dried for 24 hours. Filtered, and the filtrate was evaporated under reduced pressure, to obtain a compound of formula 3-2 as (where X = SCN).

(3) nucleophile NaSCN, phase transfer catalyst is TBAB and 18-crown-6

The compound (product of Example 1, step (a)) is represented by the formula 3-1 is dissolved in dichloromethane (20ml) was added TBAB (2.8mmol) and 18-crown -6 (2.8mmol), the NaSCN (35mmol) in water (2.0ml) was added to the reaction solution. Dropping was completed, stirring was continued for 60 minutes, the solid was removed by filtration. The filtrate was washed with water, add MgSO 4 dried for 24 hours. Filtered, and the filtrate was evaporated under reduced pressure, to obtain a compound of formula 3-2 as (where X = SCN).

(4) nucleophile as NaN 3 , phase transfer catalyst is TBAB

The compound (product of Example 1, step (a)) is represented by the formula 3-1 is dissolved in dichloromethane (20ml) was added TBAB (2.8mmol), the NaN 3 (35 mmol) in water (2.0ml) solution of was added dropwise to the reaction solution. Dropping was completed, stirring was continued for 60 minutes, the solid was removed by filtration. The filtrate was washed with water, add MgSO 4 dried for 24 hours. Filtered, and the filtrate was evaporated under reduced pressure, to obtain a compound of formula 3-2 as (where X = N 3 ).

(5) the nucleophilic reagent is KCN, the phase transfer catalyst is TBAB

The compound was dissolved in methylene chloride as in formula 3-1 (20ml), was added TBAB (2.8mmol), the KCN (35mmol) in water (2.0ml) was added dropwise to the reaction solution. Dropping was completed, stirring was continued for 60 minutes, the solid was removed by filtration. The filtrate was washed with water, add MgSO 4 dried for 24 hours. Filtered, and the filtrate was evaporated under reduced pressure to remove the solvent to give a compound as shown in Formula 3-2 (where X = CN).